Ultra-high Performance Supercritical Fluid Chromatography Research Articles

Quantitative Analysis of Prenylated Constituents in Commercial Hops Samples Using Ultrahigh-Performance Supercritical Fluid Chromatography.

The importance of hops (the flowers of Humulus lupulus) as food and an herbal remedy is reflected by a large number of analytical methods published. However, supercritical fluid chromatography, a highly efficient, rapid, and "green" separation technique, has not been considered for hops samples so far. This prompted us to establish the first supercritical fluid chromatography-based protocol for the separation, identification, and quantitation of five prenylated constituents of hops. Hulupinic acid ( 1: ), a prominent oxidation product of hop acids, three flavanones, i.e., 8-prenylnaringenin ( 2: ), 6-prenylnaringenin ( 3: ), and isoxanthohumol ( 4: ), as well as the chalcone xanthohumol ( 5: ) could be baseline separated in lessthan 5 minutes using a Viridis BEH 2-EP column (3.0 × 100 mm; 1.7 µm particle size) and a mobile phase consisting of CO2 and isopropanol. Good results regarding selectivity, accuracy (recovery rates: 85.0 - 113.1%), precision (intra-day ≤ 2.1%, inter-day ≤ 3.5%), and linearity (R2 ≥ 0.99) were obtained for both photodiode array and mass detection. The lowest detection limit at 220 nm was at 0.1 µg/mL ( 1, 3: , and 4: ), with mass detection even at 0.001 µg/mL ( 4: ). As an application example of the validated method, the five hops constituents were quantified in three dietary supplements, one herbal medicinal product, and two batches of hop flowers (Lupuli flos). In most samples analyzed, the major component was 5: (0.01 - 1.02%), whereas the major component in Lupuli flos samples was compound 1: (0.12 - 0.21%). This protocol offers a fast and environmentally friendly alternative to liquid chromatography for the quality control of hops.

Determination of 14 heterocyclic aromatic amines in meat products using solid-phase extraction and supercritical fluid chromatography coupled to triple quadrupole mass spectrometry.

A novel, simple, and sensitive method has been developed for simultaneous determination of 14 heterocyclic aromatic amines in meat product using solid-phase extraction combined with ultrahigh-performance supercritical fluid chromatography coupled to tandem quadrupole mass spectrometry. The analytes could be separated within 7 min and identified using their retention times and mass. The developed method was validated based on the linearity, limits of quantification, precision, and accuracy. The recovery ranged from 52.3 to 97.5% with an acceptable standard deviation, which is not higher than 6%. The limits of quantitation ranged from 0.03 to 0.17µg/kg. The selectivity and sensitivity were satisfactory in multiple reaction monitoring mode. The method was applied to commercial meat products, and the results demonstrated that the novel method has potential for the analysis of the targets in food matrices. This is the first work reporting thesimultaneous quantification of 14 heterocyclic aromatic amines by means of ultrahigh-performance supercritical fluid chromatography coupled to tandem quadrupole mass spectrometry.

Analysis of the Stereoisomers (cis, trans) of Polyhedral Oligomeric Silsesquioxane Compounds by UHPSFC-QTof MS

Polyhedral oligomeric silsesquioxanes (POSSs) are a class of important hybrid inorganic/organic materials. When POSSs are used as the building block in polymerization, the materials show superior performance over their organic counterparts in many areas due to the special Si–O cage structure. However, the stereoisomers (cis, trans) of polyhedral oligomeric silsesquioxane (POSS) show some differences in improving polymer property and it is difficult to get the pure stereoisomer by synthesis. Therefore, a method is required to separate and purify the mixture of POSSs. In this study, the isomers of POSS compounds and the impurities were separated by ultra-high performance supercritical fluid chromatography coupled quadrupole time-of-flight mass spectrometry (UHPSFC-QTof MS). To separate the isomers of POSS, the crucial parameters of UHPSFC-QTof MS were investigated. Under the optimal conditions, the isomers of product and impurities were separated in 4 min. On account of exact mass measurement, some impurities were identified. This is the first study on the analysis of isomers of POSS using UHPSFC-QTof MS. Based on this analysis method, it is helpful to optimize the conditions of synthesis.

Lipidomic characterization of exosomes isolated from human plasma using various mass spectrometry techniques

Ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS), ultrahigh-performance liquid chromatography - mass spectrometry (UHPLC/MS), and matrix-assisted laser desorption/ionization (MALDI) - MS techniques were used for the lipidomic characterization of exosomes isolated from human plasma. The high-throughput methods UHPSFC/MS and UHPLC/MS using a silica-based column containing sub-2 μm particles enabled the lipid class separation and the quantitation based on exogenous class internal standards in <7 minute run time. MALDI provided the complementary information on anionic lipid classes, such as sulfatides. The nontargeted analysis of 12 healthy volunteers was performed, and absolute molar concentration of 244 lipids in exosomes and 191 lipids in plasma belonging to 10 lipid classes were quantified. The statistical evaluation of data included principal component analysis, orthogonal partial least square discriminant analysis, S-plots, p-values, T-values, fold changes, false discovery rate, box plots, and correlation plots, which resulted in the information on lipid changes in exosomes in comparison to plasma. The major changes were detected in the composition of triacylglycerols, diacylglycerols, phosphatidylcholines, and lysophosphatidylcholines, whereby sphingomyelins, phosphatidylinositols, and sulfatides showed rather similar profiles in both biological matrices.

Comparative study of ultra-high-performance supercritical fluid chromatography and ultra-high-performance liquid chromatography to simultaneous determination of ten components in Radix hedysari

Background: In China, dried root of Hedysarum polybotrys Hand-Mazz, known as Hong Qi (Radix hedysari [RH]), is one of the important traditional Chinese medicines (TCMs). The long analysis time was a major limitation when high-throughput techniques are required for research or quality control purposes. Materials and Methods: It is necessary to develop a high?throughput method for the determination of multiple components of TCMs by investigating the separation characteristics of the target with ultra-high-performance liquid chromatography (UHPLC) and ultra-high-performance supercritical fluid chromatography (UHPSFC). Objective: This study intends to establish a high-throughput method for simultaneous determination of ten compounds in RH by UHPSFC and to develop a standardized method to comprehensively evaluate the quality of RH samples. In addition, this study was based on UHPLC and UHPSFC systems to understand and compare the effects of different separation parameters on ten target compounds in RH. A high-throughput and reliable solid-phase extraction method for simultaneous detection of ten target compounds in RH was established and validated. Results: The optimal analysis method was achieved using HSS C18SB column, gradient elution, flow rate of 1.5 mL/min, diode array detection at 270 nm, and back pressure at 12.41 Mpa. The separation method was validated sufficiently by examining the precision, recoveries, linearity, range, limit of detection, and limit of quantification and was successfully applied to quantify the 25 RH samples. In addition, the comparative study also demonstrated the complementary nature of the two separation modes. Conclusion: This is the first study that ten compounds in RH were separated and analyzed simultaneously with UHPSFC-diode array detector. In addition, under the separation conditions of UHPSFC and UHPLC, the comparison of chromatographic parameters shows that a combination of these two separation techniques efficiently separates the ten target compounds in RH. The results obtained in this study are useful for future TCM quality control and high-throughput analysis.

An automatic online solid-phase dehydrate extraction-ultra-high performance supercritical fluid chromatography-tandem mass spectrometry system using a dilution strategy for the screening of doping agents in human urine

An automatic online solid-phase dehydrate extraction (SPDE)-ultra-high performance supercritical fluid chromatography (UHPSFC)-MS/MS system was developed in this study, in which the automatic SPDE procedure was coupled with UHPSFC to allow UHPSFC to analyze aqueous samples directly. Moreover, a pre-column dilution strategy was employed, which focused the analytes in strong desorption solvent on the column head and helped to obtain narrow and symmetric peaks. The online SPDE-UHPSFC-MS/MS system was firstly applied to the screening of 45 prohibited substances in human urine for doping control, during which all the mechanisms and features of the online system were fully studied. The majority (91%) of the target compounds achieved weak matrix effects (80–120%), indicating that the online method was accurate and reliable thanks to the SPDE procedure and efficient UHPSFC separation. Owing to the reduction of the matrix effects, large volume injection and the pre-column dilution, the online system could achieve high sensitivity with the LODs ranging from 0.0380 ng L−1 to 1.24 μg L−1. Under the optimized conditions, the extraction recoveries of 66% target analytes were more than 50%. All the target compounds showed good linearity with linear correlation coefficients higher than 0.9928. The accuracy values of all the spiked prohibited substances were within 80.8–119.7%, while the RSDs% for the intra-/inter-day precision were within 10.8% and 15.4%. Compared with the dilute-and-shoot-ultra-high performance liquid chromatography-MS/MS method, in which the urine samples were simply diluted before analyzing, this online method was superior in sensitivity and reducing matrix effects, which demonstrated its utility in doping control. Compared with the previously reported online SPE-SFC system, the online SPDE-UHPSFC-MS/MS system showed advantages in automation, efficiency, sensitivity and chromatographic performance. In summary, the online SPDE-UHPSFC-MS/MS system is capable of analyzing complex aqueous samples.

The expression of anaerobic metabolites in sweat and sebum from human skin subjected to intermittent and continuous mechanical loading

Pressure ulcers (PUs) represent a substantial burden to both patients and healthcare providers. Accordingly, effective prevention strategies should follow early detection of PUs. Anaerobic metabolites, such as lactate and pyruvate, are promising noninvasive biomarkers indicative of tissue ischaemia, one of the major mechanisms leading to PU development. The aim of this study was to investigate if the temporal release profile of these metabolites in sweat and sebum is sensitive to detect local tissue changes resulting from prolonged mechanical loads.The sacrum of healthy volunteers was subjected to two different loading protocols. After a baseline measurement, the left and right side of the sacrum were subjected to continuous and intermittent loading regimen, respectively, at a pressure of 100 mmHg. Biomarker samples were collected every 20 min, with a total experimental time of 140 min. Sweat was collected at 37 ∘C and 80% relative humidity, and sebum at ambient conditions, from 11 to 13 volunteers, respectively. Both samples were analysed for lactate and pyruvate concentrations using ultra-high performance supercritical fluid chromatography mass spectrometry. Prior to analysis metabolite concentrations were normalized to individual baseline levels and, in the case of sweat, additional normalization was performed to an unloaded control site to account for fatigue of sweat glands.Although substantial variability was present, the temporal release profiles of both sweat and sebum metabolites reflected the applied loading regimen with increased levels upon load application, and recovery to baseline levels following load removal. Highest relative increases were 20% and 30% for sweat lactate and pyruvate, respectively, and 41% for sebum lactate. Sebum pyruvate was not present in quantifiable amounts. There was a linear correlation between the individual responses to intermittent and continuous loading.The present study revealed that metabolite biomarkers in both sweat and sebum were sensitive to the application of mechanical loads, indicative of local ischaemia within skin and soft tissues. Similar trends in metabolic biomarkers were observed in response to intermittent and continuous loading regimens in both sweat and sebum. Metabolites represent a potential means to monitor the health of loaded skin and soft tissues informing timely interventions of PU prevention.

An analytical strategy for accurate, rapid and sensitive quantitative analysis of isoflavones in traditional Chinese medicines using ultra-high performance supercritical fluid chromatography: Take Radix Puerariae as an example

Isoflavones are phenolic phytoestrogens due to their structural similarity to estradiol, so they usually serve as active component for quality control of traditional Chinese medicines (TCMs) rich in isoflavones. However, TCMs contains various kinds of similar isoflavones, especially isomers, which to a significant extent hinders accurate analysis of isoflavones in TCMs. Here, we present a novel analytical strategy for quality control of TCMs rich in isoflavones using ultra-high performance supercritical fluid chromatography coupled to photodiode array detection (UHPSFC–PDA) and tandem mass spectrometry (UHPSFC–MS/MS). Both chromatography and mass spectrometry parameters were optimized in order to develop an accurate, rapid, sensitive method for quantification of isoflavones. The reproducibility of quantitative analysis of multi-components by single marker (QAMS) using UHPSFC–PDA was discussed in terms of mobile phase gradient, temperature and backpressure for the first time. An analytical method for the analysis of isoflavones using UHPSFC–MS/MS was developed for the first time, and the established method was successfully applied to quantify isoflavones in three species of Radix Puerariae. The study showed Radix Pueraria Peduncularis contained higher amounts of isoflavones than Radix Puerariae Thomsonii, and it is worth noting that Radix Pueraria Peduncularis was often overlooked by researchers. It took less than 8 min with the current method and the limit of detection was not more than 0.05 ng/ml, which was definitely sufficient for anlysis of various samples from TCMs without enrichment.

An environmentally friendly and green method for separation and determination of eight phenolic acids in raw and processed Tussilagofarfara L. by ultra-high performance supercritical fluid chromatography

Phenolic acid as an important active ingredient of traditional Chinese medicine, have received extensive attention due to its various biological activities and pharmacological effects. However, the presence of isomers makes the separation of phenolic acids challenging. In the present study an environmentally friendly, sustainable, lower cost, and green that reduce amounts of organic solventsultra-performance convergence chromatography (UPC2) method was developed for separation and quantification eight phenolic acids. Ultra-high performance supercritical fluid chromatography combines the advantages of supercritical fluid chromatography and ultra high performance liquid chromatography technologies with shorter analysis time, higher resolution and better environmental compatibility. The effects of different chromatographic column, co-solvent, column temperature and back pressure were studied. The BEH column was provided the most suitable separation withgradient elution in 20 min using CO2 as the mobile phase and methanol/acetonitrile (70/30, v/v) with 2% TFA as the co-solvent at a flow rate of 0.4 mL/min. A better and more symmetrical peak shape were obtained. Subsequently, the established method was applied to the quantification of phenolic acids in raw and processed Tussilago farfara L. and validated according to FDA guidelines, showed good precision, accuracy, repeatability and linearity in the concentration range. Our findings confirm that the method could be used as an efficient method for exploring phenolic acids of the quality and separation of traditional Chinese medicine.

Simultaneous determination of 17 bisphenols in polycarbonate by ultra-high performance supercritical fluid chromatography with tandem mass spectrometry.

A novel method was developed for the first time for the determination of 17 bisphenols by ultra-high performance supercritical fluid chromatography with tandem mass spectrometry. Under the optimal conditions, 17 bisphenols were separated successfully on a high density diol column in 9min using methanol and carbon dioxide as mobile phase. 0.02% ammonium hydroxide/methanol v/v was used as the post-column compensation solvent to improve response of mass spectrometry. Linear relations of matrix-matched calibration curve were favorable over the selected concentration range of 1-100μg/kg with correlation coefficients greater than 0.9981. The method limit of detection and limit of quantitation were 0.1-0.5μg/kg and 0.5-2.5μg/kg, respectively. The average recoveries at three spiked levels in polycarbonate were in the range of 81.8-114.5%. Intra-day and inter-day precisions for six replicates were below 15.0%. This method was successfully applied to determine bisphenols in polycarbonate.

Comprehensive analysis and quality assessment of Herba Epimedii from multiple botanical origins based on ultra-high performance supercritical fluid chromatography coupled with quadrupole time-of-flight mass spectrometry and photodiode array detector

Herbal medicines (HMs) usually consist of complex matrices of phytochemical compounds responsible for their efficacy. Supercritical fluid chromatography (SFC) and ultra-high performance SFC (UHPSFC) are efficient techniques that can be advantageously used for the analysis of complex HMs. Here, the comprehensive UHPSFC methods were established for separation of similar flavonoids, chemical profiling and differentiation, and quality assessment of Epimedium species (EPs). An efficient separation of 12 polar flavonoids was enabled in UHPSFC with an important additive, oxalic acid. Then, 51 flavonoids were characterized, and 28 potential markers enabling the differentiation of E. wushanense from other four species were discovered by UHPSFC-QTOF/MS combined pattern recognition multivariate statistical analysis. Additionally, the UHPSFC-PDA was developed and validated for simultaneous quantification of 7 predominant flavonoids in five EPs, and remarkable variation in their contents was observed. This integrated UHPSFC approach has potential applications in systematic analysis and quality evaluation of HMs containing hydrophilic flavonoids.

Purification of drug degradation products supported by analytical and preparative supercritical fluid chromatography

A stressed degradation (oxidation) was employed to produce metabolites from an active pharmaceutical ingredient (API) with large molecular weight (about 900 g/mol). An analytical chromatographic method was desired to compare the products generated by different degradation methods while a multi-gram-scale preparative chromatographic method was necessary to purify the produced metabolites. Supercritical fluid chromatography (SFC) was selected for both tasks as no other chromatographic method had achieved the resolution of the API and metabolites (two isomeric mono-oxide species and one di-oxide). First, an analytical-scale method was developed with ultra-high performance supercritical fluid chromatography (UHPSFC). Achiral stationary phases containing sub-2 μm fully porous particles or sub-3 μm superficially porous particles, and chiral phases containing 3 and 5 μm fully porous particles were selected for a first screening with gradient elution (carbon dioxide – methanol containing additives). The stationary phase providing the most promising results was ACQUITY Torus 2-PIC (100 x 3 mm, 1.7 μm, Waters). A central composite design (CCD) was conducted to optimize the gradient program and oven temperature. Final gradient conditions were as follows: 50–70% methanol in 3.8 min with oven temperature set at 36 °C, back-pressure set at 11 MPa and flow-rate at 0.8 mL/min. The optimized method was employed to analyze samples obtained with different degradation conditions. Then the method was adapted and transferred to preparative-scale SFC on a 5 μm-particles Torus 2-PIC stationary phase (150 x 30 mm). The method was modified to comprise an isocratic step followed by a gradient, favoring peak shape of the last eluting compound and minimal volume of collected fractions. Batch injections in gradient mode were carried out to purify six grams of crude product.

Hyphenation of ultra-high performance supercritical fluid chromatography with atmospheric pressure chemical ionisation high resolution mass spectrometry: Part 2. Study of chromatographic and mass spectrometry parameters for the analysis of natural non-polar compounds

An analytical method based on Ultra-High-Performance Sub/Supercritical Fluid Chromatography (UHPSFC) coupled with High-Resolution Mass Spectrometry (HR-MS) equipped with an Atmospheric Pressure Chemical Ionisation source (APCI-Q-TOF-HRMS) was developed for the screening of various compounds in oily samples. The hyphenation was achieved using a hybrid UHPSFC system for the vegetable oil analysis, mainly composed of fatty acids, diacylglycerols, triacylglycerols but containing also some minor bioactive compounds. No split was used with this ionisation source in this optimized configuration, allowing the introduction of all compounds in the mass spectrometer, ensuring a better sensitivity. This configuration is preserving peak shapes and efficiency because the dead volume of this interface is optimized by the supplier for the hyphenation with open cell detectors such as Evaporative Light Scattering Detector (ELSD) or MS detectors. The influence of analytical conditions (UHPSFC and APCI parameters) on MS response was studied to understand the behaviour of analytes in UHPSFC-APCI-MS. The tested parameters were: the corona discharge value, the nebulising gas pressure, the drying gas flow rate, MS source temperature and the mobile phase flow rate (at constant modifier percentage). A factorial experimental design was carried out for this study, which displayed the major and negative role of increased nebulising gas pressure. The factorial design was renewed with the four remaining parameters, allowing to enlighten and explain their different effects on MS responses. Finally, some vegetable oils were analysed by UHPSFC-APCI-HRMS with these optimal conditions to determine the chemical structures of unknown compounds from oily samples.

Fast Simultaneous Determination of Eight Sudan Dyes in Chili Oil by Ultra-High-Performance Supercritical Fluid Chromatography.

A rapid and simple ultra-high-performance supercritical fluid chromatography (UHPSFC) with photodiode array (PDA) method was developed and validated for simultaneous determination of eight Sudan dyes in chili oil. In particular, a pair of isomer, Sudan red B and Sudan IV, was included in the analysis. After being diluted with dichloromethane, the analytes were separated on an Acquity UPC2 HSS C18 SB column with gradient elution using CO2 as the mobile phase and acetonitrile/methanol (v/v, 45/55, containing 0.1% formic acid) as the organic modifier. Analytes were quantified by external calibration curves over ranges of 0.5–50 mg/L, with correlation coefficients above 0.999. The method gave recoveries of the target compounds (spiked at levels of 1, 5, and 25 mg/kg) ranging from 82.6 to 108.3%, with intraday and interday relative standard deviations of less than 8.0% and 8.6%, respectively. The limits of detection (LODs) and the limits of quantification (LOQs) for eight dyes were from 0.10 to 0.30 mg/kg and 0.30–1.00 mg/kg, respectively. This method was applied for the analysis of chili oil samples collected from the supermarket in Beijing. This validated that the UHPSFC-PDA method provides a useful strategy for the simultaneous determination of Sudan dyes in chili oil for routine analysis.

Enantiospecific UPC2 SFC-MS method for separation and quantification of R &amp; S-Eliglustat tartrate in presence of its stereo isomers and degradation impurities

Abstract Eliglustat tartrate is an oral inhibitor of glucosylceramide synthase used in the treatment of Gaucher’s disease. This drug is administered as enantio-specific trans-(R,R) isomer and there is a possibility to have three more potential stereo isomers due to two chiral centers, owing to the toxicological difference between stereoisomers chiral method with high sensitivity is required to detect low level contaminations. A method for enantiomeric separation and quantification of trans-(R,R) Eliglustat tartrate in presence of other isomers trans-(S,S) Eliglustat, cis-(R,S) Eliglustat, cis-(S,R) Eliglustat was developed and validated using ultra-high performance supercritical fluid chromatography with tandem mass spectrometry (UPC2 SFC-MS) which is termed as a green chromatographic technique. Chiral separation was developed using Chiralcel OX-3 (4.6 * 150 mm) 3 µm column which has 4 chloro, 3 methyl phenyl carbamate as chiral selector, with a flow rate of 3 g/min using CO2 and 0.5% diethyl amine in ethanol: methanol (1:1) as a co-solvent. The effect on the enantioseparation by varying chiral additives, gradient conditions and content of organic modifier were investigated. The method has good linearity with concentration ranges from 0.25 to 7.5 µg mL−1. The detection limit was 0.08 µg mL−1 and quantification limit was 0.25 µg mL−1. Forced degradation studies were performed and all the degradation impurities were separated from product peak, the molecular weight of impurities were identified by using UPC2-Mass spectrometer. The method was validated in line to ICH guidelines in terms of linearity, accuracy, precision, LOQ, LOD and robustness.

Detection and Quantitation of ACCUTRACE S10, a New Fiscal Marker Used in Low-Duty Fuels, Using a Novel Ultrahigh-Performance Supercritical Fluid Chromatography–Mass Spectrometry Approach

A new ultrahigh-performance supercritical fluid chromatography–mass spectrometry (UHPSFC–MS) method has been developed using electrospray ionization (ESI) to detect and quantify a new fiscal fuel marker, ACCUTRACE S10, that was introduced into fuel in the U.K. and Ireland from April 1, 2015. S10 is synthesized by the Dow Chemical Company and is used as a replacement for ultraviolet–visible (UV–vis) fuel markers, such as quinizarin, Euromarker, and Solvent Red 24. It is UV–invisible, is doped at a low level (2.5 ppm), and was designed for detection using modern gas chromatography–mass spectrometry (GC–MS) instrumentation. All currently proposed methods for the determination of the ACCUTRACE S10 fuel marker in diesel fuel (automotive diesel oil, with current specifications of EN 590 and ASTM D975) use GC–MS, and two-dimensional (2D) GC–MS is necessary to detect the marker at tank dilutions. However, the lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for S10 are severely compromised...

Current trends in supercritical fluid chromatography.

Supercritical fluid chromatography (SFC), which employs pressurized carbon dioxide as the major component of the mobile phase, has been known for several decades but has faced a significant resurgence of interest in the recent years, thanks to the development of modern instruments to comply with current expectations in terms of robustness and sensitivity. This review is focused on the recent literature, specifically since the introduction of modern systems but in relation to older literature, to identify the changing trends in application domains. Typically, natural products, bioanalysis, food science, and environmental analyses are all strongly increasing. Together with reduced extra-column volumes in the instruments, the advent of sub-2-μm particles and superficially porous particles in the stationary phases is favoring ultra-high-performance SFC (UHPSFC) allowing for improved resolution and faster analyses, but without the constraints of viscous liquids encountered in ultra-high-performance liquid chromatography (UHPLC). Hyphenation to mass spectrometry is also more frequent and opened the way to new application domains, and raises different issues from liquid chromatography mobile phases, especially due to decompression of carbon dioxide. It is also shown that the frontiers between SFC and HPLC are fading, as switching from one method to the other, even within the course of a single analysis, is facilitated my modern instruments. The present review is not intended to be exhaustive but rather giving a snapshot of recent trends in supercritical fluid chromatography, based on the observation of about 500 papers published in English-written peer-reviewed journals from 2014 to 2018. Graphical abstract ᅟ.

Ultra-high performance supercritical fluid chromatography in impurity control: Searching for generic screening approach

In this study, 63 compounds measured as 10 individual mixtures containing in each case an active pharmaceutical ingredient and its relevant impurities, and additional set of 7 basic beta blockers were analyzed using ultra-high performance supercritical fluid chromatography with UV and mass spectrometry detection. The separations were accomplished using 8 different stationary phases (diol, diethylamine, 2-picolylamine, 1-aminoanthracene, BEH 2-ethylpyridine, BEH, CSH pentafluorophenyl, and HSS C18 SB), 6 modifiers (methanol, ethanol, isopropanol, methanol/acetonitrile, methanol/ethanol, ethanol/acetonitrile) and 5 additives in methanol (0.1% formic acid, 10 mmol/L ammonium formate, 10 mmol/L ammonium acetate, 0.4% ammonium hydroxide, and 2% water).The resulting chromatograms were evaluated using 6 selected parameters: number of eluted peaks, number of separated peaks, resolution between active pharmaceutical ingredient and following impurity, peak symmetry, peak width at 50% of the peak height, and selectivity. Volatile additives such as ammonium formate, ammonium acetate, and especially ammonium hydroxide provided overall generic approach with very good chromatographic performance. Combined modifier methanol/acetonitrile (1:1) was important for separations of some critical pairs in case of neutral compounds. Diol stationary phase proved its suitability for wide range of applications when the separations of all mixtures were satisfactory on this column, except for vardenafil and beta blockers mixture. HSS C18 SB stationary phase offered unique complementary selectivity especially for analysis of structurally close compounds and/or isomers.

Determination of Five Retinol Isomers in Animal Livers Using Ultra-High Performance Supercritical Fluid Chromatography

A novel and efficient ultra-high performance supercritical fluid chromatography method was developed for the quantitative analysis of five retinol isomers in animal livers. The separation of the five retinol isomers was carried out using an Acquity UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 µm) with acetonitrile as a co-solvent. By optimizing the columns, gradient program, co-solvent, column temperature and backpressure, the five retinol isomers and the internal standard 11-cis-13,14-dihydroretinol were successfully separated within 20 min. Samples were saponified and extracted by solid-supported liquid–liquid extraction using a diatomaceous earth cartridge. Comparing with the traditional liquid–liquid extraction, the extraction enables the reduction of time-consuming and laborious procedures. This method used 11-cis-13,14-dihydroretinol as an internal standard to improve the precision and accuracy of quantitative analysis. The correlation coefficients (r2) of the calibration curves were all above 0.999, the limits of detection for the five retinol isomers were in the range of 0.10–0.20 µg mL− 1, and the limits of quantification were in the range of 0.33–0.66 µg mL− 1. The mean recoveries were from 92.5 to 102.5%. The interday and intraday relative standard deviations were within 10%. This method was successfully applied to the determination of retinol isomers in ten raw animal livers and animal liver products (chicken, duck, pig, cattle, and sheep).

Development and validation of ultra-high performance supercritical fluid chromatography method for quantitative determination of nine sunscreens in cosmetic samples

A fast and simple utra-high performance supercritical fluid chromatography (UHPSFC) method has been developed for the determination of nine sunscreens (UV filters), namely 2-ethylhexyl-2-hydroxybenzoate (ES), ethylhexyl-methoxycinnamate (EMC), benzophenone-3 (BZ3), octocrylene (OCR), bis-ethylhexyloxyphenol-methoxyphenyl triazine (EMT), butyl-methoxydibenzoyl-methane (BDM), diethylamino-hydroxybenzoyl-hexyl-benzoate (DHB), ethylhexyl-triazone (ET), and diethylhexyl-butamido-triazone (DBT) in cosmetic samples. The separation was achieved with Acquity UPC2 Torus 2-PIC (100 × 3.0 mm, 1.7 μm) column. The influence of key chromatographic parameters on resolution was evaluated. The optimal mobile phase was a gradient mixture of carbon dioxide and methanol containing ammonium acetate, at flow rate 1.2 mL min−1. The back-pressure of the system was set to 150 bar and the temperature to 40 °C. The compounds were determined with diode-array detection at 280 nm, 305 nm and 340 nm depending on absorbance maxima. The proposed UHPSFC method provided separation of the nine target sunscreens within 2.5 min without labor-consuming sample pretreatment procedure. The method was validated according to the concept of the total error and the accuracy profile. The inter-laboratory reproducibility was evaluated between two independent laboratories (in France and Czech Republic). The reliability of the developed method was shown by application to commercial sunscreen-containing cosmetic sample obtained from French market. The measured limits of quantification showed the suitability of the proposed method for determination of UV filters considering the European Union requirements.